Nanofluidics for biomolecular separations
thesisposted on 29.03.2022, 01:51 authored by Michael Andrei Startsev
Nanofluidics, microfluidics and lab-on-a-chip technology has garnered much attention in recent years due to the opportunities it offers in analytical chemistry and point-of-care. However, an understanding of the physicochemical phenomena associated with biomolecular separations within nanofluidic devices is needed to enable those possibilities. The work presented in this manuscript describes a novel nanofluidic device designed to simultaneously separate and concentrate dilute biomarkers within low volume samples. The mechanism of separation driving biomolecules within the nanofluidic device is the continuous counteracting forces of electrophoresis and electro-osmosis in the presence of a spatial gradient that modulates molecular mobility (conductivity gradient or pH gradient) induced along the nanochannels of the device. R-phycoerythrin, streptavidin-Dylight, DNA, and PNA (peptide nucleic acid) were focused and separated within the nanochannel under various conditions and their respective behaviours were characterized. A pH gradient was applied along the nanochannel device by introducing two different pH buffers at opposite terminals of the nanochannel and applying an axial voltage across the channel. Fluorescent proteins, phycoerythrin and Dylight labelled streptavidin, were concentrated within the channel reaching concentration enhancement (CE) factors of over 380 within 5 minutes. A conductivity gradient was also applied along the nanochannel by introducing buffers of dissimilar salt concentrations at the terminals of the nanochannel in order to create a trapping condition similar to the pH gradient method. Simultaneous separation and concentration of Bacillus cereus DNA and R-phycoerythrin was achieved to a CE of over 900. The conductivity gradient technique was also used to detect hybridization between oligonucleotides and complementary strands of PNA. This work illustrates how these electrofocusing devices are capable of matrix-free separation and concentration of biomarkers. In the future, this technology may enable on-chip integration of orthogonal separation techniques with mass spectrometry.